Code for the RNA-Seq and ChIP-seq analyses used for the paper entitled: The H2A.J histone variant contributes to Interferon-Stimulated Gene expression in senescence by its weak interaction with H1 and the derepression of repeated DNA sequences
| Main Authors: | MANGELINCK, Adèle, MANN, Carl |
|---|---|
| Format: | info software Journal |
| Bahasa: | eng |
| Terbitan: |
, 2020
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| Subjects: | |
| Online Access: |
https://zenodo.org/record/4147010 |
Daftar Isi:
- Abstract for overall study: The histone variant H2A.J was previously shown to accumulate in senescent human fibroblasts with persistent DNA damage to promote inflammatory gene expression, but its mechanism of action was unknown. We show that H2A.J accumulation contributes to weakening the association of histone H1 to chromatin and increasing its turnover. Decreased H1 in senescence is correlated with increased expression of some repeated DNA sequences, increased expression of STAT/IRF transcription factors, and transcriptional activation of Interferon-Stimulated Genes (ISGs). The H2A.J-specific Val-11 moderates the transcriptional activity of H2A.J, and H2A.J-specific Ser-123 can be phosphorylated in response to DNA damage with potentiation of its transcriptional activity by the phospho-mimetic S123E mutation. Our work demonstrates the functional importance of H2A.J-specific residues and potential mechanisms for its function in promoting inflammatory gene expression in senescence. Specific description of ChIP-seq: Our biochemical experiments suggested that increased expression of H2A.J in senescent fibroblasts could contribute to the decrease of histone H1 in senescence. Previous work from the Jordan lab has shown that H1 depletion can lead to the derepression of Interferon-Stimulated Genes (ISGs). Our previous work showed that H2A.J depletion inhibited inflammatory gene expression in senescent fibroblasts, and ectopic expression of H2A.J in proliferating fibroblasts stimulated inflammatory gene expression, and in particular, ISGs. Thus, decreased H1 levels in senescent fibroblasts, dependent in part on increased levels of H2A.J in senescence, could contribute to the transcription of ISGs as part of the inflammatory gene expression program in senescence. To test this possibility further, we performed genome-wide analyses of the activating histone H3-K4me3 activation mark and RNA pol II occupancy in WI-38/sh-NT and WI-38/sh-H2AFJ in proliferating and senescent fibroblasts. We induced senescence with etoposide using a protocol that allowed rapid induction of inflammatory genes. Macs2 was used to identify ChIP-seq peaks that distinguished the sh-NT and sh-H2AFJ fibroblasts in etoposide-induced senescence. Peaks with differential intensity were mapped to proximal genes with ChIPseeker, and the generated gene set was then analyzed by Reactome pathway enrichment analysis. Interferon signaling pathways and Interferon-Stimulated Genes were the highest scoring pathways. Notably, MIR3142HG encoding mIR-146A, was the gene whose H3-K4me3 peak was the most affected by H2A.J knock-down Description of small-RNA-seq: RNA was purified using the mirVana miRNA Isolation Kit (ThermoFisher Scientific, #AM1560) according to the manufacturer’s protocols to yield a fraction containing total RNAs and a second fraction enriched in small RNAs. The total RNA fraction was used with the Illumina Ribo-Zero Scriptseq library kit to deplete cytosolic rRNA and prepare DNA librairies. The small RNA fraction was used with the NEBNext small RNA library prep kit (New England Biolabs, #E7300) to produce a small RNA library. Libraries were subjected to size selection on a 6% native polyacrylamide gel to enrich for molecules with inserts within the miRNA size range (18-25 nt). Both librairies were sequenced using 43 bp paired-end reads. RNA-seq of small RNAs showed that mIR-146A was highly induced in senescent fibroblasts as has been previously reported. Consistent with the decreased levels of the activating H3-K4me3, miR-146A levels were greatly reduced in H2A.J knock-down cells relative to the NT controls. Interestingly, most previous data on miR-146A indicate that it is a negative feedback regulator of the Interferon pathway (39, 40), thereby suggesting a defect in this pathway in cells depleted for H2A.J. Description of Ribo-Zero RNA-Seq: H1 depletion was associated with transcriptional derepression of repeated DNA sequences and transcriptional activation of ISGs. We previously reported transcriptome analyses of WI-38/hTERT fibroblasts expressing sh-NoTarget and two different H2AFJ-shRNAs using Illumina Bead Cheap arrays that showed a defect in inflammatory gene expression in H2A.J-deleted fibroblasts in senescence. We now describe Ribo-Zero RNA-seq to more globally assess effects of H2A.J depletion on RNA levels in senescence. Principal component analysis showed good separation of senescent versus proliferating and sh-NT versus sh-H2AF transcriptomes. Comparison of sh-NT cells induced into senescence with etoposide relative to proliferating cells showed the anticipated repression of E2F-activated cell cycle genes and loss of canonical histone gene expression accompanied by increased expression of NFkB and Interferon-regulated inflammatory genes. Comparison of sh-H2AFJ cells in senescence relative to sh-NT control cells in senescence also confirmed the previous microarray analysis indicating a defect in inflammatory gene expression in H2A.J knock-down cells. GSEA indicated that down-regulation of Interferon Response Genes was the most significantly affected gene set, which is consistent with the CHIP-seq results for H3-K4me3 and RNA polII. We next compared levels of RNAs transcribed from repeated DNA sequences. Consistent with previous studies, most differentially expressed repeat RNAs were up-regulated in senescent relative to proliferating cells with the greatest increase assigned to HSATII pericentromeric satellite sequences. Comparing H2A.J-ko cells with sh-NT cells in senescence, we found lower levels of RNAs transcribed from 7 repeated DNA elements and increased transcription from 4 elements. Three repeated DNA elements, MER53, LTR26, and LTR23, were induced during the senescence of sh-NT control cells and underexpressed in the H2A.J knock-down cells in senescence relative to sh-NT cells.
- BioRxiv preprint: doi: https://doi.org/10.1101/2020.10.29.361204